NLRs represent the intracellular analog to the TLRs (Toll-like receptors), important mediators of innate immunity. NLRs contain a nucleotide-binding fold called NACHT, plus variable numbers of Leucine-Rich Repeat (LRR) domains, often combined with either PYRIN domains (PYDs) or Caspase-associated recruitment domains (CARDs). Oligomerized NLRs serve as platforms for recruiting signaling proteins brought to the complex via PYD, CARD, or other domains, including inflammatory Caspases involved in cytokine processing and apoptosis. These proteins complexes have been dubbed, inflammasomes. This renewal application focuses on NLR-family member NALP1 as a prototype of the NLR family. During the previous funding period, we reported that the LRRs of NALP1 are required for responses to microbial ligands, analogous to the LRRs of TLRs in sensing pathogens. We also demonstrated that the LRRs inhibit the NACHT domain from binding nucleotide triphosphates (NTPs) and oligomerizing, until stimulated by microbial ligands. We also learned that activation of NALP1 is inhibited by members of the Bcl-2 family, proteins involved in apoptosis regulation. Recently, we also obtained evidence that some homologs of Bcl-2 encoded in poxviruses bind members of the NLR family, suggesting a mechanism by which they suppress host inflammatory responses. Moreover, polymorphisms in NALP1 have recently been linked to differential sensitivity to bacterial virulence factors, including anthrax toxin (Lethal Factor [LF]), a zinc metalloproteinase that depends on NALP1 for inducing Caspase-dependent killing of macrophages, thereby impairing host defense. Our goals for this competitive renewal application are to address the following questions about NLR-family proteins, focusing on NALP1 as a prototype: (1) What is the mechanism by which bacterial ligands activate the NALP1 inflammasome?; (2) What is the mechanism by anthrax toxin [LF] activates NALP1?; (3) What is the mechanism by which Bcl-2 and Bcl-XL suppress NALP1 activation?; and (4) What are the effects of viral Bcl-2 homologs on NALP1? Answers to these questions will provide insights into the mechanisms regulating NLR-family proteins, laying a foundation for addressing unmet medical needs in the areas of autoimmunity, allergy, inflammation, vaccines, and infectious diseases, while also aiding in development of counter measures for bioterrorism. PROJECT NARRATIVE NLR proteins constitute a large group of molecules involved in host-defense, protecting our body from bacteria and viruses as a first-line of defense, known as innate immunity. Imbalances in the endogenous mechanisms that regulate NLR-family proteins are associated with inflammatory and autoimmune diseases, where these proteins become hyperactive. Conversely, susceptibility to infection is associated with insufficient or inappropriate activity of these proteins. NLRs are attacked by proteins encoded in the genomes of bacteria and viruses, thereby thwarting immune responses. The bacterial and viral pathogens known to attack NLRs include agents of bioterrorism, including Anthrax Toxin and poxviruses. This proposal seeks to improve understanding of the mechanisms regulating NLRs, including the endogenous cellular mechanisms and the mechanisms relevant to bacterial and viral infection. The proposed project is relevant to several diseases with unmet medical needs, including autoimmunty, chronic inflammation, allergy, infection, and vaccines.